Project Summary/Abstract Genetic and acquired defects in Ca release channels, ryanodine receptors (RyR2s), underlie a spectrum of lethal cardiac disorders ranging from arrhythmias to heart failure (i.e. ryanopathies). Although RyR2 is considered to be a logical target for the treatment of these disorders, effective therapies based on normalization of RyR2 function are lacking. This is in part due to the complex nature of RyR2 regulation. In this proposal, we will test the hypothesis that intelligently-engineered proteins coupled with AAV-mediated gene transfer provide a strategy for the rational design of therapies to treat ryanopathies. Based on calmodulin's (CaM) role in regulating RyR2, we propose a novel cardiac gene therapy approach against ryanopathies using RyR2-specific and multi-target CaMs, i.e therapeutic CaMs (TCaMs). This strategy is based on the concept suggested by our initial studies that various regulators of RyR2 function converge on a common mechanism of refractoriness that controls RyR2 activity dynamics and Ca signaling stability during the cardiac cycle. Genetic and acquired RyR2 defects, including mutations in RyR2, CASQ2 and CaM, alter RyR2 refractoriness resulting in disturbed Ca cycling, premature aberrant Ca release and consequent arrhythmias. Therapeutic CaMs (TCaMs) designed to reset RyR2 refractoriness, regardless of the underlying etiology, will provide a general treatment strategy for ryanopathies. To accomplish this goal, we will use multi-scale studies (from molecule to whole animal) that combine the expertise of a protein biochemistry (Davis) lab and a cellular physiology (Gyorke) lab using novel and cutting edge protein delivery approaches for engineered CaMs and state-of-the art multi-compartmental and multicellular imaging tools for testing their functional effects. Additionally, studies using genetic mouse models and preparations from a clinically relevant canine heart failure (HF) model will provide a ?proof-of-principle? for new therapeutic strategies based on desynchronization of aberrant Ca release by CaM-mediated slowing of Ca signaling refractoriness. We propose to: 1) Test the hypothesis that different genetic forms of CPVT impair RyR2 function through a common mechanism: shortening Ca signaling refractoriness; and 2) Engineer ?therapeutic?(T)CaMs for the treatment of ryanopathies.